Saccharomyces bromocresol green screen

Eureka, I would like to share my latest plating results with you. You might know that I am very interested in isolating any kind of wild yeasts from commercial sour beers. The most difficult task in this whole isolation process is to differentiate normal Saccharomyces cerevisiae colonies from other yeast species such as Brettanomyces.

Previous studies to develop new kind of agar media to detect Brettanomyces in wine samples showed bromocresol green to be a useful indicator to detect acid producing Brettanomyces strains [Rodrigues et al., 2001; Couto et al., 2005, EP 1185686 A1]. In this case, bromocresol green acts as a pH-indicator and turns yellow in the presence of acid which is produced by some Brettanomyces species. The authors further added cycloheximide to the media to prevent any growth of Saccharomyces. Concluding from the previously cited publications an addition of cycloheximide to agar media should already be enough to differentiate between Saccharomyces and Brettanomyces colonies by simply inhibiting the growth of Saccharomyces. Further antibiotics could be added to prevent the growth of bacteria.

Other studies showed that bromocresol green alone can be used to differentiate between the two yeasts in absence of antibiotics [Yakobson, 2010]. In addition, bromocresol can diffuse into yeast colonies and form green colonies due to the accumulation of the dye [Yakobson, 2010]. However, some Brettanomyces strains seem to be able to form white colonies again. This has been shown in other experiments as well [Rodriguez, 2012; BKYeast, 2012]. Yakobson mentions that the dye gets actively metabolized by Brettanomyces and hence the white colonies again. Unfortunately, I could not find any source investigating how exactly the dye get metabolized. Yakobson further mentions that some Saccharomyces strains can form white colonies as well which would make it even more difficult to differentiate between Saccharomyces and Brettanomyces.

The aim of this study was to screen different Saccharomyces strains for their ability to form white colonies on bromocresol green containing agar.

Material

• Sabouraud agar 4% glucose, Art. X932.1, Roth
• Bromocresol green sodium salt, Art. KK18.1, Roth
• Saccharomyces strains from Wyeast and White Labs

19 different Saccharomyces strains including one Saccharomyces mixture (WY3056) were plated on Sabouraud agar containing bromocresol green. Bromocresol green was added as aqueous, sterilized solution to the sterilized Sabouraud agar until the agar turned blue. The plates were incubated at room temperature at a dark place until colonies were visible. A control was included (no yeast streaked) to observe any color changes of the agar due to environmental effects (photo bleaching, oxidation, decay etc). The following yeast strains were used for this screen.

Number	Product name
WY1010	American Wheat
WY1056	American Ale
WY1084	Irish Ale
WY1728	Scottish Ale
WY1762	Belgian Abbey II
WY2112	California Lager
WY2278	Czech Lager
WY2487	Helle Bock
WY3056	Bavarian Wheat Blend
WY3068	Weihenstephan
WY3333	German Wheat
WY3522	Belgian Ardennes
WY3638	Bavarian Wheat
WY3711	French Saison
WY3726	Farmhouse Ale
WY3864	Canadian/Belgian Ale
WY3942	Belgian Wheat
WY3944	Belgian Wit
WLP002	English Ale
Control	N/A

Results Part 1

Colonies were visible after four days of incubation (Fig 1-5). The control showed no colony formation and the agar showed no color change. The colors of the agar were compared with the control.

Fig 1: Yeasts on bromocresol agar after four days. Left: WY1762, Top: WY3522, Right: WY3864; Bottom: Control

Fig 2: Yeasts on bromocresol agar after four days. Left: WY3942, Top: WY3944, Right: WY3726; Bottom: WY3711

Fig 3: Yeasts on bromocresol agar after four days. Left: WY2487, Top: WY2112, Right: WY2278; Bottom: WY1010

Fig 4: Yeasts on bromocresol agar after four days. Left: WY3638, Top: WY3068, Right: WY3333; Bottom: WY3056

Fig 5: Yeasts on bromocresol agar after four days. Left: WLP002, Top: WY1056, Right: WY1728; Bottom: WY1084

Some Saccharomyces strains were able to change the color of the agar from green-blue to yellow. Only two yeast strains, WY3333 German Wheat and WY3726 Farmhouse Ale, grew as white colonies on bromocresol green agar after four days. This already is proof that some strains indeed can grow as white colonies on bromocresol green. The plates were further incubated and after a total of twelve days, the color of the colonies were evaluated for a second time (Fig 6-10). Sorry for the bad quality of the pictures.

Fig 6: Yeasts on bromocresol agar after 12 days. Left: WY1762, Bottom: WY3522, Right: WY3864; Top: Control

Fig 7: Yeasts on bromocresol agar after 12 days. Left: WY3942, Top: WY3944, Right: WY3726; Bottom: WY3711

Fig 8: Yeasts on bromocresol agar after 12 days. Left: WY2487, Top: WY2112, Right: WY2278; Bottom: WY1010

Fig 9: Yeasts on bromocresol agar after 12 days. Left: WY3638, Top: WY3068, Right: WY3333; Bottom: WY3056

Fig 10: Yeasts on bromocresol agar after 12 days. Left: WLP002, Top: WY1056, Right: WY1728; Bottom: WY1084

One could observe that some of the colonies now have white edges and a green centre. All these colonies were still counted as green colonies.

This time less yeast strains turned the agar to a yellow color because the control agar lost a lot of its blue color. The global decrease of the blue color in the agar might originate from diffusion of acids secreted by yeasts that turned the agar yellow. Or due to the diffusion of the dye into the colonies. Further on to the white yeast colonies. WY3333 German Wheat and WY3726 Farmhouse Ale still grew in white colonies. In addition, WY3864 Canadian/Belgian Ale and WLP002 English Ale now grew as white colonies as well. One might expect further yeast strains to form white colonies with a prolonged incubation time because a lot of the colonies already have white edges and a remaining green centre.

After 17 days of incubation, the colonies looked as shown below (Fig 11).

Fig 11: Yeasts on bromocresol agar after 17 days. Left/bottom: WY2487, Left/Top: WY2112, Right/Top: WY2278; Right/Bottom: WY1010

A lot of yeast colonies now turned into white colonies as expected (Fig 12). The color was now evaluated by looking at the entire colonies visible for a particular strain. If more than 50% of the colonies were white, the yeast was counted as white. Like the WY2278 Czech Lager shown in Fig 11. On the other hand, all the other yeasts shown in Fig 11 were counted as green like the WY3711 French Saison in Fig 12.

Fig 12:WY3711 French Saison colonies after 17 days of incubation

After 17 days of incubation, only seven out of the 19 screened yeasts still had green colonies. All the other ones turned white in the meantime. To put it in numbers. After 4 days 2/19, after 12 days 4/19 and after 17 days of incubation 12/19 yeast strains formed white colonies (Fig 13). This clearly shows a time dependency.

Fig 13: Yeast screen results on heavily stained bromocresol green agar

Results Part 2

The blue color in the agar plates (Fig 1-5) was quite heavy and to test whether a lower concentration of bromocresol green in the agar leads to the same results as discussed above, a second experiment was conducting by streaking the exact same yeast strains on some Sabouraud agar containing bromocresol green. This time a lower concentration of bromocresol green was used.

Quantification of the color after three days of incubation (Fig 14-18):

Fig 14: Yeasts on bromocresol agar after three days. Left: WY3864, Top: Control, Right: WY1762; Bottom: WY3522

Fig 15: Yeasts on bromocresol agar after three days. Left: WY3942, Top: WY3944, Right: WY3726; Bottom: WY3711

Fig 16: Yeasts on bromocresol agar after three days. Left: WY2112, Top: WY2278, Right: WY1010; Bottom: WY2487

Fig 17: Yeasts on bromocresol agar after three days. Left: WY3638, Top: WY3068, Right: WY3333; Bottom: WY3056

Fig 18: Yeasts on bromocresol agar after three days. Left: WLP002, Top: WY1056, Right: WY1728; Bottom: WY1084

Yet again some colonies grew as white colonies and others grew as green ones (Fig 19). Comparing the results with the one concluded from the first experiment, WY3726 Farmhouse Ale, WLP002 English and WY3864 Canadian/Belgian Ale showed white colonies. In contradiction with the first experiment are the color morphologies of WY3333 German Wheat, WY1728 Scottish Ale and WY3711 French Saison. WY3333 grew as white colonies in the first experiment and as green ones in the second one. On the other hand, WY3711 and WY1728 grew as white colonies in the second experiment.

Fig 19: Closer look at Fig 15

The colours were again determined after further incubation. Agar plates shown after 12 days of incubation (Fig 20-24).

Fig 20: Yeasts on bromocresol agar after 12 days. Left: WY3864, Top: Control, Right: WY1762; Bottom: WY3522

Fig 21: Yeasts on bromocresol agar after 12 days. Left: WY3942, Top: WY3944, Right: WY3726; Bottom: WY3711

Fig 22: Yeasts on bromocresol agar after 12 days. Left: WY2112, Top: WY2278, Right: WY1010; Bottom: WY2487

Fig 23: Yeasts on bromocresol agar after 12 days. Left: WY3638, Top: WY3068, Right: WY3333; Bottom: WY3056

Fig 24: Yeasts on bromocresol agar after 12 days. Left: WLP002, Top: WY1056, Right: WY1728; Bottom: WY1084

Twelve days of incubation and all the yeast strains have the same color like a few days ago. The plates were further incubated and a final color determination was conducted after 17 days (not shown).

The results of the second run are summarized in Fig 25. WY1010 American Wheat, WY1084 Irish Ale, WY1762 Belgian Abbey II, WY2112 California Lager, WY2278 Czech Lager, WY3068 Weihenstephan and WY3942 Belgian Wheat all had white colonies after 17 days (Fig 25). 13/19 yeast strains grew as white colonies after 17 days of incubation (Fig 25).

Fig 25: Yeast screen results on light-stained bromocresol green agar

Discussion

Comparing the two experiments, some strains such as WY1010 American Wheat, WY1728 Scottish Ale, WY2112 California Lager, WY3711 French Saison and WY3942 Belgian Wheat only grew in white colonies after 17 days on the light stained agar media and not the heavy stained one (Fig 26). WY3056 Bavarian Wheat Blend, WY3522 Belgian Ardennes and WY3638 Bavarian Wheat grew as white colonies on heavily stained agar but as green ones on lightly stained agar media (Fig 26). This might be an indicator that the bromocresol green concentration might influence the color change as well.

Fig 26: Differences between the two experiments

As a general trend, the different yeast strains seem to form white colonies after further incubation. However, two strains (WY3333 German Wheat and WY3726 Farmhouse Ale) grew on heavily stained agar as white colonies from very early on (Fig 13) and four additional ones on lightly stained agar (Fig 25). Yakobson states on his website that Wit yeasts can metabolize bromocresol green (http://www.brettanomycesproject.com/2009/03/wln-agar-medium/). In this screen the Wit strain from Wyeast (WY3944) did not grew as white colonies in both experiments (Fig 13, 25). Not even after 17 days of incubation.

Some words about the color of the agar media. Fig 1 to 5 are nice examples to show that the color of the bromocresol containing media changes its color from green to yellow. In both experiments, the color of the control agar turned to a yellow color as well. The plates were stored at a dark place to prevent any influence of light (photobleaching effects). The change in color might be due to secretion of acids (bromocresol changes color at lower pH to yellow), due to a take-up of the dye by the yeast cells like stated by Yakobson in case of Brettanomyces. Another possibility might be the stability of bromocresol green itself. If one imagines bromocresol green to be a relatively unstable molecule, the loss of the green color might be due to the depletion of the dye. Yakobson further mentions that Brettanomyces can even metabolize the dye and therefore grow as white colonies. All the cells not able to metabolize the dye remain as green colonies. Unfortunately, I could not find any evidence for this statement showing that Brettanomyces really metabolize the dye. Nor any evidence that Saccharomyces can do it. Maybe the cytoplasm of Brettanomyces cells have a lower pH and therefore turn the dye from green to yellow. There might even be some truth about this hypothesis since some Brettanomyces strains are known to secrete acetic acid under aerobic conditions. It is therefore not clear to me why/how the colonies turn from green to yellow.

I would like to discuss bromocresol green as a useful tool to differentiate between Brettanomyces and Saccharomyces. Although I did not show any Brettanomyces colonies here, the bromocresol screen strongly suggests that some Saccharomyces strains can grow as white colonies on bromocresol green containing agar media. This makes a differentiation already a bit harder. In addition, a majority of Saccharomyces yeast strains appear as white colonies after a longer incubation period. BKYeast came to the conclusion that differentiation based on bromocresol green might only be possible in a short time frame in mixed cultures (Saccharomyces and Brettanomyces grow on the same plate). The results from these experiments show that even in pure cultures, and in absence of Brettanomyces, a lot of the Saccharomyces strains tested turned from green to white within a short period of time. All these results strongly suggest that any differentiation solely based on bromocresol green might only be useful in a short period of time.

Summary

Bromocresol screen is a widely used differentiating dye to differentiate between Saccharomyces and Brettanomyces. Brettanomyces known for their capability to grow as white colonies while Saccharomyces grow as white ones. It has been reported that some Saccharomyces strains grow as white colonies as well and therefore making a differentiation more difficult [Yakobson, 2010]. Screening different Saccharomyces cerevisiae strains on bromocresol green containing Sabouraud agar revealed some strains capable of growing as white colonies from the very beginning where the majority of yeast strains grew as green ones. Therefore showing that indeed some yeast strains can grow as white colonies. After further incubation, the majority of the yeast strains turned from green to white coloured colonies. There seems to be a general trend for Saccharomyces cerevisiae strains to form white colonies after extended incubation times. However the reason for this observation is not clear at this point as well as the mechanism leading to the observed change in color. It can’t be excluded that different sources like instability of bromocresol green itself or any environmental factor lets the colonies turn from green to yellow.

Due to these observations, bromocresol green as a tool to differentiate between Brettanomyces (known to grow as white colonies) and Saccharomyces might only work within a small time frame. This has been previously observed by BKYeast as well.

Outlook

Brettanomyces bromocresol green screen similar to the one shown here for Saccharomyces. In addition, try to grow Brettanomyces anaerobically to test whether the colonies grow as white or green ones (acid theory mentioned in the discussion).

References:

• Couto J.A., Barbosa A. and Hogg T. (2005) A simple cultural method for the presumptive detection of the yeasts Brettanomyces/Dekkera in wines. Letters in Applied Microbiology, 41, 505-510
• BKYeast (2012) http://bkyeast.wordpress.com/2012/02/19/selective-media-part-i/
• EP 1185686 A1, Culture medium for detection of Dekkera and Brettanomyces, http://www.google.com/patents/EP1185686A1?cl=en
• Rodrigues N., Goncalves G., Pereira-da-Silva S., Malfeito-Ferreira M. and Loureiro V. (2001) Development and use of a new medium to detect yeasts
  of the genera Dekkera/Brettanomyces. Journal of Applied Microbiology, 90, 1-12
• Rodriguez J. (2012) Brooklyn Brewery Samples on New Wild Yeast Media, sciencebrewer.com
• Yakobson C. (2010) Brettanomyces project, http://www.brettanomycesproject.com/2009/03/wln-agar-medium/

I am open to any discussions and feedback concerning this experiment. Thank you for reading.

Beer Candy Syrup Experiment

Eureka, today’s post is all about a recent candy syrup experiment. Making my own candy syrup was very tempting but I never got to actually do it. This all changed a few days ago when we decided to put the ingredients together for a long-planned batch of Belgian Dubbel. I used Brewferm’s Candy syrup (180 – 220 EBC) before and was quite pleased what this syrup contributes to a beer. In my opinion, the most important factor why Westlveteren 12, Rochefort 10 and all the other great Belgian Quadrupel beers are so amazing is because of the candy syrup they use. Candy syrup can contribute a lot of the dark fruit character, pear aroma, toffee and caramel one can taste in Quadrupels. And we wanted to have some of this character in our next Dubbel. However, our supplier was out of the Brewferm Candy Syrup and this gave us the opportunity to make our own candy syrup.

I don’t want to go into the chemical details here since I haven’t put much thoughts into that. To make candy syrup one needs sugar, a liquid and a nitrogen source for the Maillard reactions. Yeast nutrients are a very common to supply the needed nitrogen. We made three batches of candy syrup: One with plain table sugar, one batch with raw cane sugar and one batch with plain table sugar again but with beer instead of water. The process however was the same for all three batches and is explained in more detail in pictures below. All the recipes are based on J. Smiths Belgian Candy Sugar experiment aired by Basic Brewing Radio on May, 7th 2009. Every batch started with 0.45 kg of sugar (1 lbs), 0.5 cup of liquid and 1.5 table spoons of yeast nutrients. We added the sugar and yeast nutrients to the cooking pot first and afterwards the liquid. Not stirred at all. The temperature was then slowly raised to approximately 143°C (290°F). Again not stirred once during the whole process.

Fig 1: Table sugar based candy syrup at 75°C (167°F)

At 75°C (167°F) the first bubbles could be observed (Fig 1). At 85°C (185°F) a very distinctive ammonia smell escaped the pot.

Fig 2: Table sugar based candy syrup (20 min)

20 min after the first bubbles arose, the color of the candy syrup slowly turned darker (Fig 2). The temperature was around 127°C (260°F).

Fig 3: Table sugar based candy syrup

The color now got darker really fast as the temperature got closer to 143°C (290°F) (Fig 3). As the temperature reached 143°C (290°F), we added roughly 0.8 cup of water. Even for the share made with beer. This worked well for the table sugar based syrup but the raw sugar and beer based syrup foamed a lot. The addition of the water has to be done really carefully! One can easily get burned.

Fig 4: Table sugar based candy syrup after a second water addition

The temperature was then raised to 116°C (240°F) again (Fig 4) and the hot syrup was then transferred into glass jars (Fig 5).

Fig 5: First batch of candy syrup in glass jars

This is the basic process how we did our candy syrup. The second batch was the same as the first one but we used raw cane sugar instead of white table sugar. Just to see if there is a difference between different sugar sources. During the first batch we decided to use beer instead of water. Again just for fun and to see how it turns out. Beer should have extra nitrogen and maybe increase the flavors of the candy syrup as well.

Fig 6: Beer based candy syrup

We decided to use a heavy #36 Rusalka Russian Imperial Stout for that purpose (Fig 6). Added 0.5 cup of Imperial Stout and made another batch of candy syrup as described above. There was a minor disadvantage with this candy syrup already, it foamed a lot (Fig 7). One had to be really careful to prevent an over boil and we had to stir once in a while.

Fig 7: Beer based candy syrup foams a lot…

Another disadvantage was that the whole mixture was dark already. Therefore no chance to observe any color changes at all. A blind flight. But the previous two batches gave us enough courage to simply follow the temperatures and add water at 143°C (290°F). Then re-heating the mixture to 116°C (240°F) and then fill the syrup in glass jars.

After the syrups cooled down, a first tasting (we could not wait to try the syrups).

Let me begin with the reference, the Brewferm Candy Sugar we used up to now. This is a very brown syrup with a relatively low viscosity. Much like liquid honey. The syrup smells like pears, figs and raisins. On the palate again pears, figs and some caramel. Even some minor toffee character. Baseline set.

Next, the table sugar based candy syrup. This syrup is already darker than the reference and very sticky and way more viscous. Faint aroma (couldn’t detect anything). Huge caramel, some toffee and pears again on the palate. Less sweet as well. And beside the caramel and toffee, less intense than the reference.

Moving on to the raw sugar candy syrup. Darker again with a nice red shine to it. Very viscous again but less viscous than the table sugar syrup. More intense aroma than table sugar syrup but we could not put the aroma into words. It smells fruity and maybe even some decent smoke, wood character. On the palate huge pear aroma, some bitterness and burnt character. Some toffee, caramel and coffee? Really hard to describe.

At last to the Imperial Stout based syrup. Really nervous. This is like the experiment where one could expect either a complete disaster or something unique. Lets see. Similar viscosity as the reference. Huge chocolate aroma, smells like a milk coffee and some malt character as well. The character of the beer seems to overpower the smell of the syrup. Palate: Pear, figs, chocolate, no roast character, not bitter, cold coffee. Wow! Way more character than the previous three syrups. Even a spicy character in there and some wood. Simply amazing.

Comparing all the four syrups, the most interesting one is definitely the one made with beer. It even out-rules the reference syrup. Actually, we were not expecting that at all. Our goal was to make some candy syrup to see how the process works and maybe find a decent homemade-syrup substitute for the Dubbel. In our opinion, both the table sugar and raw sugar candy syrup could work as substitutes for the Brewferm candy syrup. However, the Brewferm syrup still tastes a bit better. This is not true for the beer syrup. Simply something unique. Not only has the beer increased the overall flavors of the syrup but the beer contributed some of its own character to the syrup as well. This opens a lot of opportunities for making new varieties of beer based candy syrups. Not only could we experience that the base sugar makes a difference but even the liquid does as well.

Thanks to our supplier who could not supply us with the candy syrup. Would one still call this a supplier?!? We might have never came up with this beer-based candy syrup idea after all. Let me know if anyone else has tried this before. Thanks for reading and commenting, stay tuned!

#23P Single Wheat Experimental Batch (SWaSH)

Eureka, today’s post is about another SMaSH (single malt and single hop) experiment. The reason for doing this recipe was to find out how a single wheat malt beer would taste like and to find out whether it is possible to brew a 100% wheat beer in the first place. I therefore call this kind of beer SWaSH for single wheat and single hop. One problem with such a setup is the fact that wheat malt does not have husks as most of the barley malts. These husks are necessary for lautering with a perforated bottom to get a kind of filter bed. Without such husks one could imagine that the wheat malt plugs the perforated bottom and makes a lautering relatively hard or even impossible. However, I have no experience with a 100% wheat malt based beer and a perforated bottom. I chose to do a brew in a bag instead to circumvent the lautering with my perforated bottom. That’s basically the most exciting part abput this recipe:

Recipe:	SWaSH
Numbers:	Volume [L]	5 (1.32 gal)
	Original gravity	12°P
	Terminal gravity	4.8°P
	Color	Around 4 EBC
	IBU	10 IBU
	ABV	4%
Grains:	Wheat malt (4 EBC)	1 kg
Hops:	Tettnanger (4.2% AA)	3 g and boil for 60 min
	Tettnanger (4.2% AA)	2.5 g and boil for 30 min
Yeast:	Wyeast’s	#3068 Weihenstephan
Water:	Burgdorf	Mash: 2.5 L (0.66 gal), sparge: 4 L (1.1 gal) @78°C (172°F)
Rest:		Mash in @47°C (117°F), 45 min @45°C (113°F), 15 min @53°C (127°F), 30 min @63°C (145°F), 40 min @72°C (162°F), 10 min @ 78°C (172°F)
Boil:		Total 60 min
Fermentation:	Primary	5 days @20°C (68°F) in plastic fermenter
	Secondary	N/A
Maturation:	Carbonation (CO2 vol)	4 with sugar addition (40 g sugar to 4.5 L beer)
	Maturation time	14 days

02/26/2011: Brew day. I chose to do a small SWaSH batch with 1 kg of wheat malt and some Tettnanger hops. I then preheated the mash water up to 47°C and inserted the malt bag into the kettle and added the crushed wheat malt. I then did the rests as mentioned in the recipe and pulled the bag out after resting at 78°C for 10 min and washed the grains with the sparing water. By the way, the iodine test was negative. Boiled the wort for 60 min with the small hop additions and cooled the wort down to 20°C (68°F) and added 25 billion (2.5E10) yeast cells for 5 L of wort.

03/05/2011: Its bottling time. Five days after brew day I bottled the beer with some sugar to get a carbonation level of approximately 4.0 vol of carbon dioxide. I then left the bottles carbonate and mature for nearly 14 days at room temperature and then put the bottles in a refrigerator.

05/07/2011: The beer is now nearly two months in the bottles and its time for a tasting. I tasted the beer before but never did any tasting notes. I kind of forget to do tasting notes and when I do them most of the beers are already past their best-to-drink date…

Aroma: Lots of banana, no hop character and no malt character detectable. Smells like a traditional South German wheat beer.

Appearance: Pale yellow color, lots of carbon dioxide bubbles rise to the top and form a very creamy, white head. Very good head resistance. I added some of the yeast sediment to get the real wheat character.

Flavor: Not a lot is going on here. Banana again and some of the lightly sour, bread, grainy character of the wheat malt is detectable. And the yeast gives the beer the typical yeasty, doughy character.

Mouthfeel: Light body, lively carbonation. Rather short and sweet, malty aftertaste. Very refreshing.

Overall Impression: The beer is still fresh and very refreshing after some time in the bottle. Lots of the characteristic wheat beer character such as banana and the character you get from the wheat malt are present as well. Sure a well made beer. However, it is a bit boring…

It seems that a 100% wheat beer is possible to make and enough enzymes are present in the wheat malt for a complete mash conversion. What about the wheat malt? First of all, the beer tasted like expected. Rather one-dimensional and lots of the wheat malt comes through. Hops are in the background and some of the wheat yeast character comes through as well. That’s what I was going for in the first place with this experiment. At least I now know what the wheat malt can contribute to such a beer.

Comparing the flavor profile of this beer with other homemade wheat beers from the past and some commercial examples, going the extra mile with a 100% wheat malt beer does not seem to have a big impact on the beer. I therefore can’t see any advantages brewing a 100% wheat malt based beer compared to a wheat beer made with 50% wheat malt and 50% barley malt. Thanks for reading and commenting if you like and stay tuned!

Isolating the bugs from Cantillon Gueuze 2007

Fig 1: Cantillon’s Lou Pepe 2007 Gueuze

Eureka, this is another post concerning wild yeast isolation from a commercial beer. Today’s beer is Cantillon’s Lou Pepe 2007 Gueuze. I got a bottle of this particular Gueuze a year ago and stored it for another year in my cellar. By the end of June 2012, I finally got the opportunity to open the bottle and taste it.

Before heading into the tasting notes, let me give you some background information about the beer. The label on the bottle says: “Our Lou Pepe beers are all exceptional products. We only use the finest lambic to make these beers. The Lou Pepe Gueuze is a blending of only 2 years old lambics. Beer with tasteevolution. Best before 12/2029″ (Fig 2). It comes in 0.75 L bottles and 5 ABV. Bottled on the 12th of October in 2009.

Smell: Very funky and a lot of horse blanket, leather and barnyard

Taste: Very light sourness, pretty dry on the palate, grainy. Some lemon and wood notes as well. Rather nice sourness (no vinegar). Subtle notes of funkiness.

Appearance: Pours in a golden-yellow color, clear and a pretty nice white head. Not very long lasting head though. Very fizzy. Looks like a champagne

Mouthfeel: Light body, average carbonation, dry and astringent aftertaste. Some bitterness is there as well

Overall: Not bad and very easy drinkable. Not a very sour and complex Gueuze compared to others. However, a good example for the style. My rating: 80/100. I expected this beer to be more complex and less astringent.

Fig 2: Bottle description

I then streaked some of the bottle’s sediment on some Sabouraud agar plates and left the plates at room temperature for approximately three weeks until colonies were visible. I could observe two different kinds of colonies (Fig 3).

Fig 3: Cantillon’s Lou Pepe 2007 sediments on Sabouraud agar

The most colonies were similar to the whitish colonies marked as 2 in Fig 3. And there were some darker colonies (light beige) marked as 1 in Fig 3. Nearly two years after bottling the Gueuze there are still some living organisms in the bottle. The morphology of these colonies is very similar to other Brettanomyces I isolated before. I expect these colonies (marked 2) to be Brettanomyces. On the other hand, I have no clue what the microorganisms in colony 1 could be since the color is very different from Brettanomyces or Saccharomyces colonies. Maybe the micrographs give me further information? Next step was to do some microscopy observations of the two different colonies. Lets begin with the colonies marked 2 in Fig 3.

Fig 4: Micrograph of colony 2 (see Fig 3)

Fig 5: Micrograph of colony 2 (see Fig 3)

To me, the colonies shown in Fig 4 and 5 look like a kind of wild yeast. At least no Saccharomyces cerevisiae for sure. Or any other kind of bacteria due to the size of these cells. I expect these cells to be Brettanomyces due to the elongated shape of the cells and other characteristics. The cells could be Kloeckera apiculata, Pichia membranaefaciens or Hansenula… The list is not complete here. Further investigations are necessary. What about the other colonies?

Fig 6: Micrograph of colony 1 (see Fig 3)

Fig 7: Micrograph of colony 1 (see Fig 3)

First of all, the cells shown in Fig 6 and 7 look very different from the ones shown in Fig 4 and 5. These cells here are mostly circular and look very similar to Saccharomyces cerevisiae cells. Aggregation of Saccharomyces cerevisiae as it can be seen on the upper left corner in Fig 7 can be observed in wheat yeast samples as well. For me theses cells look like typical Saccharomyces cerevisiae cells although some cells seem to have a more elongated form as well. I will have to do further investigations to get more information about the cells in colony 1. Wouldn’t it be cool to have isolated some Saccharomyces cerevisiae yeast cells from an old Gueuze from Cantillon?

To summarize, I could isolate two different kinds of yeasts from a Cantillon Gueuze bottled in 2009. I have a strong feeling the cells from colony 2 belong to the specie of Brettanomyces. This is just a feeling. The strains go into my library as B05 (colony 2) and Y03 (colony 1). Further investigations are necessary to differentiate the two different strains. Cool stuff. The only verified conclusion here is: It is possible to isolate some yeasts from a Gueuze that is nearly two years in the bottle. Stay tuned for further yeast related posts!

Dry hopping vs. bitterness

Eureka, its time for another experiment. But not about yeast this time… Sorry! The following experiment is all about hops and their role during dry hopping and bitterness. All started with a batch of Pale Ale where I tried the first wort hopping technique and added some hops for dry hopping (100 g of 14% AA Simcoe to 22 L (5.8 gal)). And the tasting revealed an overpowering bitterness. And some really grassy notes as well. This made me wonder how the bitterness could increase to such a high level in the first place. And there are two possible explanations in my opinion: The first wort hopping technique or the dry hopping. And this experiment was to find out, if the dry hopping can increase the bitterness in some way. Some say that the bitterness only increases when the hops are boiled and the alpha acids isomerize. Others say that the bitterness can increase as well if the hops are not boiled. Lets find out how is right.

What I did was the following: I took a Vodka and diluted it down to have 400 mL of a 5% alcohol solution. I did so to have the same amount of alcohol like you would have it in an average beer. Using pure Vodka could lead to false conclusions since the higher alcohol content could extract different/less/more compounds from the hops. I then added 100 mL of the diluted Vodka to a bottle each and added some Simcoe hops to have the following hops to volume ratio: 4 g L^-1, 8 g L^-1, 16 g L^-1 and 32 g L^-1. Common rates for dry hopping are in the range of 3.9- to 7.8 g L^-1 (according to Calagione Sam mentioned in “Extreme brewing”).

03/02/12: Added 14% alpha acid Simcoe hops to the four bottles according to the mass to volume ratios mentioned above. I stored the bottles at room temperature at a dark place for nearly three weeks before a tasting.

03/22/12: The tasting begun. I first start with the color differences of the liquids. It could be easily observed that a higher amount of hops lead to a darker, more orange pronounced color (Fig 1). Unfortunately, there is a light difference between the two bottles shown in Fig 1 (brighter on the left side). Nevertheless, there was a difference.

Fig 1: Hop infused Vodka, 4 g hops per liter (left), 8 g per liter (right)

Then the tasting. I have to mention first, this was by far the worst tasting ever! I advice you not to replicate these results. It was just horrible. But step by step:

4 g L^-1: At the lower end of the dry hopping rates. Alcohol was easy detectable, very faint hop aroma. Maybe some hints of oranges. And the taste was just bitter. Not overpowering, but bitter.

8 g L^-1: At the higher end of dry hopping rates. More intense hop aroma than the previous one. Very grassy. And the taste was just horrible. Just like eating a hop pellet. And it was definitely more bitter than the previous one as well.

16 g L^-1: Extreme hoping rate 1. Orange notes in the aroma. Floral notes as well. This one is my favorite of the four concerning the aroma. And the bitterness was comparable with the one before.

32 g L^-1: Extreme hoping rate 2. Floral notes, very aromatic hop aroma. The aroma is too intense for my taste. And again, the bitterness is very similar to the one before.

To conclude, the intensity of the aroma increases with the amount of the hops you add for dry hopping. I guess this is no surprise. What I am a bit more surprised is the fact, that the bitterness increases with the amount of hops as well. But only to a level of 8 g per liter of liquid. Further addition of hops does not increase the bitterness. Or at least not in a way that I could taste it. I have to mention here that I am talking about perceived bitterness not IBUs. I have no idea about the IBUs of the four samples.

Lets assume the IBUs of the different beers are the same if we consider that the IBUs originate from isomerized hops. Lets further assume that no isomerization happened during the experiment. How could we explain the increase in bitterness now? One possibility could be the extraction of compounds from the hops by the alcohol present. This would imply that further compound(s) in the hops can increase the bittering sensation. And basically lead to the conclusion that a higher amount of dry hops can increase the bitterness. Not the IBUs but the perceived bitterness.

I assumed that hops for dry hopping could maybe just add a bit of bitterness. But here we are talking about huge impacts. The liquids with the highest amounts of hops tasted like liquid hop pellets… I am still not really convinced here. Maybe this is just something happening only in the used Vodka-based liquids. It is therefore advisable to replicate these results using a fermented barley-based liquid. However, I conclude that a higher amount of hops in a Vodka-based liquid leads to an increasing perceived bitterness (can’t tell if the IBUs increased).

Please let me know if anyone out there has an idea what happened here or if you obtained different/same results as well. Cheers and stay tuned for further post.

Isolating the bugs from Trois Dames Oud Bruin

Eureka, it has been a while since the last post about isolating some wild bugs from a commercial beer. Luckily, there are still some beers in my cellar with bugs I would like to isolate (Orval, Lost Abbey, BFM, Cantillon, Jolly Pumpkin…). The beer I am talking about today is from a Swiss brewery called Les Trois Dames (The three women). This brewery produces an Oud Bruin (Flanders Brown) made with apricot wine. The beer comes with an ABV of 7.2%. Maybe some of my tasting notes first.

Aroma: Sour and funky smell. Pretty awesome! Could detect some cherry notes. In my opinion one of the best smelling beers in Switzerland…

Appearance: Red-brown color, clear appearance, 1 finger tan head with some carbonation. Nothing special here.

Flavor: Now begins the fun part… Unfortunately, the complexity of this beer is very limited. I could detect some sour cherry notes. The sourness is just right. Not too overpowering. However, nothing else. Not even a hint of apricots…

Mouthfeel: Light body, average carbonation level, pretty dry and sour finish.

Overall Impression: There is the right amount sourness in this beer and the aroma of this beer promises a lot. Unfortunately, the flavors speak another language. The beer has a very limited complexity in my opinion. Maybe the beer was too young? Only a second tasting could tell. To summarize, this is a pretty nice beer and because this is a beer made from a Swiss brewery, it is a winner for me for sure. I would give this beer a rating of 85 out of 100. I am a bit disappointed about the lacking apricot notes. I should get myself a bottle of apricot wine one day and compare the apricot flavors there. Maybe the apricot flavors are already very subtle in the apricot wine?

Anyway, this post is about the bugs in the beer. What I did is already a standardized technique for me to isolate some bugs from such a beer. I made myself a small DME (dry malt extract) starter and dumped the dregs of the beer in there and left it for some days. Approximately a month later, I plated some of the starter liquid on some Sabouraud agar plates and waited… Eleven days later, there were some colonies visible on the agar plate (Fig 2).

Fig 2: Sabouraud agar with colonies of Les Trois Dames Oud Bruin bugs after eleven days

The colonies morphology: White, not glossy, wavy margin, convex elevation, circular and the plate had a hint of an acetic acid smell. The acetic acid smell already made me wonder what I got myself here. I then picked a single colony and had a look at it with my microscope.

Fig 3: Les Trois Dames Oud Bruin bugs

Fig 4: Les Trois Dames Oud Bruin bugs

First of all, due to the size of the cells, I assume that all the cells visible in the following pictures are yeasts. I could observe some oval formed cells and some with the mysterious vacuole (Fig 3). And yet again, some cells which adhere to others (Fig 3). For me some of the cells in Fig 3 look like normal brewers yeast (Saccharomyces cerevisiae) and others don’t. Then again, I could observe some elongated cells (Fig 4). I already have an idea about these cells but will have to do further research first. Lets move on to the picture at a higher resolution.

Fig 5: Les Trois Dames Oud Bruin bugs

There are these dark spots again which I could observe in Brettanomyces bruxellensis before (Fig 5). My conclusion from the observations. I assume that I got myself some Brettanomyces bruxellensis and maybe some other Brettanomyces species. Some Saccharomyces cerevisiae could be in there as well. I guess this is all I can tell you about the bugs right now without doing any sophisticated lab tests. Nevertheless, I once again could show that it is possible to harvest some wild yeasts from a bottle. My next steps concerning wild yeasts will be further investigations about the elongated cells as it can be seen in Fig 4. There is a hypothesis in my head what these cells might be and I already found some promising evidence for my hypothesis. Another running project is to isolate the bugs from a Cantillon Kriek. Will post about the results for sure. By the way, the starter I made with the dregs from the Les Trois Dames bugs had no pellicle. Stay tuned for further yeast ranching experiments. Cheers!